Procaspase 3

Whole-cell protein lysates were prepared and Western blotting was carried out as previously described^{49 (link)}. PARP, FAS, DR5, BIM, BID, NOXA, PUMA, and procaspase-3-specific antibodies were obtained from Cell Signaling Technology (Danvers, MA). Cullin-3-specific antibody was obtained from BD Biosciences (Santa Jose, CA); p53- and p21-specific antibodies were obtained from Santa Cruz Technologies (Dallas, TX); FLIP-specific antibody (NF6) was obtained from Adipogen (San Diego, CA). Caspase-8 antibody was from Enzo Life Sciences (Farmingdale, NY). Secondary horseradish peroxidase-conjugated antibodies from Cell Signaling Technology (Danvers, MA) were used for detection on a G-Box digital developer (Syngene Cambridge, UK). Antibody catalog numbers are listed in Supplementary Table S4.

Total cell protein was isolated using RIPA buffer (Thermo Scientific, PI-89900) and quantified using BCA Protein Assay Kit (Pierce Biotechnology, 23225). Protein samples were run through SDS-PAGE and were transferred to polyvinylidene fluoride membrane, followed by a block in 10% dried non-fat milk, and then probed with primary antibodies against Caspase-3 (Novus Biological, 9662S), Procaspase-3 (Cell Signaling, 9662S), LC3B (Novus Biological, NB100-2220), SQSTM1/p62 (Thermo Scientific, H00008878-M01), LAMP-2 (Santa Cruz, sc-18822), ATG9A (ab108338), TFEB (Cell Signaling, 4240), GAPDH (Millipore, MAB374) and corresponding HRP conjugated goat anti-rabbit (Santa Cruz, sc-2004), goat anti-mouse (Bio-Rad, 172-1011), or donkey anti-goat (Santa Cruz, sc-2020) secondary antibodies. Proteins were visualized and quantified using chemiluminescent detection kit (GE Healthcare, Amersham ECL) and exposed to X-ray film (Thermo Scientific, CL-X Posure Film) or captured on an Amersham Imager 600 (GE Healthcare, CCD Model). The intensity for each band was densitometrically quantified and normalized against loading control using ImageJ software.

Total protein (equal amounts) were separated by SDS-PAGE, transferred onto Immobilon®-P Transfer Membrane (Millipore Corporation, Billerica, MA, USA) and blocked with 5% non-fat milk. The membranes were incubated with the respective primary antibodies against Mmp2 (Santa Cruz, CA, USA), Mmp9 (Santa Cruz, CA, USA), Bcl-2 (Santa Cruz, CA, USA), Bax (Santa Cruz, CA, USA), pro-caspase-3 (Cell Signaling Technology, Danvers, MA, United States), cleaved-caspase-3 (Cell Signaling Technology, Danvers, MA, United States), caspase-9 (Santa Cruz, CA, USA) and β-actin (Santa Cruz, CA, USA), and then incubated with the appropriate concentrations of horseradish peroxidase-conjugated secondary antibody. The blots were visualized using the SuperSignal West Pico Chemiluminescent Substrate® (Thermo Scientific, Rockford, IL, USA).

Western blotting (WB) was performed as described previously [22 (link)]. Briefly, total protein was extracted from the cells. Aliquots of total protein (10 μg) were electrophoresed on sodium dodecyl sulfate–polyacrylamide and 12% Tris–HCL gels (Bio-Rad Laboratories, CA, USA). The separated proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories) and incubated with primary antibodies overnight at 4 °C. The membranes were probed with the following antibodies: cyclin D1, p27 Kip1, CDK2, p18 INK 4C, CDK6, cyclin D3, p21 Waf1/Cip1, CDK4, pIκBα, IκBα, pro caspase 3, Ikaros, CK2α, c-Myc (1:1000 dilution; Cell Signaling Technology, Inc., MA, USA), and anti-β actin (1:3000 dilution; Sigma, Tokyo, Japan). Bound primary antibodies were detected using secondary antibodies (Cell Signaling Technology, Inc.) in a 1:10,000 dilution for anti-β actin and 1:2000 dilution for the other antibodies.

Monoclonal antibodies against PKM2, STAT1, p-STAT1, pro-caspase-3, cleaved caspase-3, and Mcl-1 were purchased from Cell Signaling Technology (Boston, MA, USA).
Cells were collected, washed with PBS and placed into 1 × Radio-Immunoprecipitation Assay (RIPA) buffer (Thermo Fisher Scientific, Waltham, MA, USA). After lysing the cells for 45 min on ice, supernatants were collected through centrifugation at 16,000 × g, 4 °C for 10 min and boiled for 10 min with loading buffer (Epienzyme, Shanghai, China). Total protein (25 μg), as measured by the bicinchoninic acid method, was separated on 10% polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes of 0.45 µm pore size. The membranes were blocked and incubated with primary antibodies diluted at a ratio of 1:1000 overnight at 4 °C. Next, the membranes were washed with 1 × Tris-buffered saline with Tween 20 (TBST) 3 times and incubated with horseradish peroxidase (HRP)-linked secondary antibodies for 1 h at room temperature. Specific bands were observed by enhanced chemiluminescence (ECL).

Human lung squamous carcinoma cell line SK-MES-1 was purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China). Isoalantolactone was purchased from the Chinese materials research center (Beijing, China), dissolved in Dimethyl Sulfoxide (DMSO), which was purchased from Shenggong Company (Shanghai, China). Fetal bovine serum (FBS) was purchased from Gibco (Carlsbad, CA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT), Hoechst 33342, Dulbecco's Modified Eagle's Medium (DMEM) and Rhodamine 123 mitochondrial specific fluorescent dye were purchased from Sigma (St. Louis, MO, USA). Cell Cycle Analysis Kit (PI+Rnase A), Annexin V-FITC Apoptosis Detection Kit, Reactive Oxygen Species Assay Kit and BCA Protein Assay Kit were purchased from Keygene Company (Nanjing, China). Polyclonal antibodies against β-actin(1:2000 dilution #4967S), Bax(1:1000 dilution #2772S), Bcl-2(1:1000 dilution #2876S), pro-caspase-3(1:1000 dilution #9662P), poly AIsoalantolactone-ribose polymerase (PARP) (1:1000 dilution #9542S), pRb(1:1000 dilution #9306S), p27(1:1000 dilution #2552), p53(1:1000 dilution #9282S), and horseradish peroxidase-conjugated secondary antibodies (goat-anti rabbit, mouse) were purchased from Cell Signaling Technology, Inc. (Shanghai, China). Western Blotting detection kit was purchased from Milipore (Billerica, MA, USA).